Protein chaperones not requiring ATP for their functioning include the Pap-D-like periplasmic chaperones found in bacteria. These proteins are able to direct formation of the appropriate three dimensional conformation in bacterial pilus and non-pilus subunits. As used herein, and unless expressly stated otherwise, the term “chaperone” means periplasmic chaperones having the distinguishing characteristics described herein.
In bacterial species, these chaperones are responsible for mediating the synthesis of large scale oligomeric structures such as pili, the adhesive fibers expressed in most bacteria of the Enterobacteriaceae family (e.g., Escherichia coli).
Pili are heteropolymeric structures that are composed of several different structural proteins required for pilus assembly. Pili, also called fimbriae or fibrillae, facilitate the adhesive qualities of bacteria that often lead to colonization and infection of various tissues of the host animal, especially on mucosal surfaces. Such adhesion is facilitated by the presence in the pilus of a protein called an “adhesin,” of which FimH is an example.
Different types of pili have been recognized. Type 1 pili-carrying bacteria recognize and bind to D-mannose in glycolipids and glycoproteins of bladder epithelial cells. Proteins forming the pili have been considered good candidates for vaccines. P pili are adhesive organelles encoded by eleven genes in the pap (pilus associated with pyelonephritis) gene cluster found on the chromosome of uropathogenic strains of E. coli. The biogenesis of P pili and Type 1 pili occurs via the highly conserved chaperone/usher pathway. (Thanassi et al, Curr. Op. Microbiol. 1,223 (1998); Hung et al, EMBO J. 15, 3792 (1994).
Type 1 pili are composite fibers consisting of a short thin tip fibrillum joined to a thicker, rigid pilus rod and comprising an ordered array of homologous pilins (FimA, FimF, FimH, and FimG) with the FimH adhesin at its tip. FimH mediates binding to mannose-oligosaccharides present on mucosal surfaces and thus mediates adherence to mannosylated receptors on the bladder epithelium, which is critical to the ability of uropathogenic Escherichia coli to cause cystitis. (See: Langermann et al, Science 276, 607 (1997).
The PapD-like superfamily of periplasmic chaperones directs the assembly of over 30 diverse adhesive surface organelles that mediate the attachment of many different pathogenic bacteria to host tissues, a critical early step in the development of disease. (See Soto and Hultgren, J. Bacteriol. 181, 1059 (1999)) PapD, the prototypical chaperone, is necessary for the assembly of P pili (Lindberg et al, J. Bacteriol. 171, 6052 (1989)) whereas its homologue, called FimC, directs the assembly of type 1 pili (Jones et al, Proc. Natl. Acad. Sci. USA 90, 8397 (1993)).
E. coli is the most common pathogen of the urinary tract, accounting for greater than 85% of cases of asymptomatic bacteriuria, acute cystitis and acute pyelonephritis, as well as greater than 60% of recurrent cystitis, and at least 35% of recurrent pyelonephritis infections. Because of the high incidence, continued persistence, and significant expense associated with E. coli urinary tract infections, there is a need for a prophylactic vaccine to reduce susceptibility to this disease. It is widely accepted that colonization of the urinary epithelium is a required early step so that disruption or prevention of pilus-mediated attachment of E. coli to urinary epithelia should prevent or retard the development of urinary tract infections.
A major drawback to adhesin based vaccines of any kind has been the fact that adhesins are often only a minor component of the pilus, cannot be produced in large quantities, and therefore will tend not to elicit a particularly strong immunogenic effect. Although recombinant technology has succeeded in producing adhesin proteins in pure form, these are often rapidly proteolytically degraded when the corresponding chaperone is absent. Such adhesins are readily stabilized by the presence of periplasmic chaperone molecules (the latter also being important in proper synthesis of adhesins).
In gram negative bacteria, such as E. coli, between the inner and outer membrane lies the periplasmic space. Proteins destined for secretion or assembly across the outer membrane often must fold within the periplasmic space prior to their secretion and/or assembly. Chaperones are often to be found within this periplasmic space. Among the proteins found in the periplasm are the adhesin FimH and its chaperone FimC.
Thus, gram negative bacteria, including many pathogenic organisms, assemble a variety of pilus and non-pilus organelles on their surfaces by the conserved chaperone-usher pathway. Many of these organelles mediate attachment to host tissues, essential for the disease process in many bacterial infections.
Throughout this disclosure the terms pilus, pili, fimbrium, fimbriae, fibrillum and fibrilla are be used interchangeably, with incidental use of the singular or plural form of any of these terms in no way limiting the breadth of the disclosed invention.
A “periplasmic chaperone” is defined herein as a protein localized in the periplasm of bacteria that is capable of forming complexes with a variety of proteins, especially pilus-proteins, including adhesins, especially FimH (where the corresponding chaperone is FimC) via recognition of a common binding epitope (or epitopes). Such chaperones are characterized by their similarity in properties to PapD, especially by their possession of an immunoglobulin-like fold for binding to pilus-proteins, such as adhesins. Such periplasmic chaperones have an effector function, specifically targeting the subunits to outer membrane assembly sites for their incorporation into pili and are characterized in part by the presence of an immunoglobulin-like fold. Like PapD, FimC uses its immunoglobulin-like domains to recognize and bind to pilus subunit proteins, such as the adhesin FimH.
The co-ordinated assembly of pili, as well as of other complex hetero-oligomeric organelles, requires correct incorporation of individual subunits in a predefined order during biogenesis and the prevention of premature associations between the intrinsically aggregative subunits. Type 1 pilus biogenesis proceeds via a highly conserved pathway that is involved in the assembly of over 30 adhesive organelles assembled by the adhesin-usher pathway in gram-negative bacteria. [Soto & Hultgren, J. Bacteriol. 181, 1059 (1999)].
While the utility of adhesins as vaccines has been demonstrated, large scale production of adhesins and other pilus-derived proteins has been complicated by the requirement of a chaperone that must be co-expressed with the adhesin in order for it to properly fold and result in a stable structure. It has now been shown that polypeptides, such as adhesins, can be prepared in a pure form without the need of co-expressing the chaperone, and without the need for the chaperone, or any other protein, thereby permitting large scale production of pure adhesins, or any other pilus subunits, for use, inter alla, as vaccines. [See: U.S. provisional patent application Nos. 60/144359, filed Jul. 16, 1999, and 60/184442, filed Feb. 23, 2000, and U.S. application Ser. No. 09/615,846, filed Jul. 13, 2000, the disclosures of which are hereby incorporated by reference in their entirety].
Pilus subunits also possess an N-terminal extension, usually about 8-20 amino acids long. In the PapD-PapK structure, this N-terminal extension is disordered. It does not contribute to the fold of the subunit but instead projects away from the subunit, where it is free to interact with another subunit. Biochemical and mutagensis experiments indicate that both the subunit groove and N-terminal extensions are involved in subunit-subunit interactions. Thus, it has been proposed that during pilus assembly, via a mechanism termed “donor strand exchange,” the N-terminal extension of a subunit replaces the G1 b-strand of the chaperone bound to its neighboring subunit. The mature pilus thus consists of an arrangement of subunits such that each contributes a strand to complete the Ig fold of its neighbor. The adhesin lacks this N-terminal extension (instead, having a receptor-binding domain N-terminal to its pilin domain) consistent with its position at the tip of the pilus, where it has no neighboring subunit that requires completion of an Ig fold.
Thus, the contribution of a chaperone, such as FimC or PapD, to the overall structure of a pilin, such as in the FimC-FimH complex, or in the PapD-PapK complex, was determined by solving the structure of such complexes by X-ray diffraction [see: Choudhury et al, X-ray Structure of the FimC-FimH Chaperone-Adhesin Complex from Uropathogenic E. coli, Science 285, 1061 (1999); Sauer et al, Structural Basis of Chaperone Function and Pilus Biogenesis, Science 285, 1058 (1999); Barnhart et al., PapD-like Chaperones Provide the Missing Information for Folding of Pilin Proteins, Proc. Natl. Acad. Sci. USA, 10, 1073/pnas.130183897 (published online Jun. 20, 2000), the disclosures of all of which references are hereby incorporated by reference in their entirety].
Such donor-strand complemented subunits as discussed in these references, especially that of Bamhart et al, as well as the structures disclosed herein, are referred to herein as “dsc-subunits,” for example, as a “dsc-pilin” or “dsc-adhesin” such as “dsc-FimH” or “dsc-PapG.” These dsc-subunits have a missing b-strand, for example (see FIG. 1 for domain structure of an adhesin, such as FimH), derived from the N-terminal extension of another subunit, linked to the C-terminus of said subunit, such as by a short amino acid linker. This missing b strand, when replaced by a donor strand sequence, thereby allows the dsc-subunit to fold into a native-like conformation in the absence of the chaperone. The dsc-subunits are thus stable, unlike wild type counterparts present in the periplasm. In accordance with the present invention, such stabilized subunits are linked to effector molecules, such as polypeptides, including antibodies, thereby providing highly useful therapeutic agents.
In accordance with the present invention, there are provided herein antibacterial therapeutics, comprising subunits linked to other polypeptide or non-polypeptide moieties, that limit or prevent attachment of bacterial pathogens and thus eliminate mortality and morbidity associated with such diseases.